The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe–4S] cluster in Escherichia coli lipoyl synthase

Abstract

The lipoyl cofactor plays an integral role in several essential biological processes. The last step in its de novo biosynthetic pathway, the attachment of two sulfur atoms at C6 and C8 of an n-octanoyllysyl chain, is catalyzed by lipoyl synthase (LipA), a member of the radical SAM superfamily. In addition to the [4Fe-4S] cluster common to all radical SAM enzymes, LipA contains a second [4Fe-4S] auxiliary cluster, which is sacrificed during catalysis to supply the requisite sulfur atoms, rendering the protein inactive for further turnovers. Recently, it was shown that the Fe-S cluster carrier protein NfuA from Escherichia coli can regenerate the auxiliary cluster of E. coli LipA after each turnover, but the molecular mechanism is incompletely understood. Herein, using protein-protein interaction and kinetic assays as well as sitedirected mutagenesis, we provide further insight into the mechanism of NfuA-mediated cluster regeneration. In particular, we show that the N-terminal A-type domain of E. coli NfuA is essential for its tight interaction with LipA. Further, we demonstrate that NfuA from Mycobacterium tuberculosis can also regenerate the auxiliary cluster of E. coli LipA. However, an Nfu protein from Staphylococcus aureus, which lacks the A-type domain, was severely diminished in facilitating cluster regeneration. Of note, addition of the N-terminal domain of E. coli NfuA to S. aureus Nfu, fully restored cluster-regenerating activity. These results expand our understanding of the newly discovered mechanism by which the auxiliary cluster of LipA is restored after each turnover.

Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

“…Surprisingly, human NFU was unable to serve as an Fe–S cluster donor to LIAS. NFU has been implicated in MMDS1 because natural mutations promote the condition, while bacterial NFU has been reported to serve as a cluster donor to LIAS in E. coli [ 8 , 18 , 29 ]. Most likely, the mutations in NFU that give rise to MMDS prevent interaction with upstream partner proteins that are involved in trafficking an Fe–S cluster to the downstream LIAS target.…”

Structural Evidence for a [4Fe‐5S] Intermediate in the Non‐Redox Desulfuration of Thiouracil

“…Such mechanism, which implies that the cluster is partly destroyed and repaired during catalysis, and thus that the enzyme cycles between a [4Fe‐3S] state and an active [4Fe‐4S] state (Scheme 1 A), has been proposed for biotin or lipoate synthesis, catalyzed by the BioB and LipA enzymes, respectively [7–10] . In contrast, for thiouridine or thiocytidine synthesis during tRNA modification, catalyzed by TtuA [12–14] and TtcA, [11] respectively, we and others proposed a mechanism in which the cluster remains intact during catalysis and proceeds via binding of an “external” S atom to one of the 4 Fe atoms, leading to a putative [4Fe‐5S] cluster intermediate from which the activated S atom is then transferred to the substrate (Scheme 1 B).…”

“…[2][3][4][5] The general agreement is that, due to its toxicity,sulfur is stored in the cell in the form of l-cysteine.I nm ost cases,p yridoxalphosphate-dependent l-cysteine desulfurases liberate sulfur from l-cysteine in the form of protein-bound persulfides which, through trans-persulfuration reactions,p rovide the sulfur atoms to the final biosynthetic enzyme. [6] However,an emerging class of sulfur transferases was recently shown to depend on ac atalytically active [4Fe-4S] cluster for redox reactions,asinthe case of lipoate and biotin synthases [7][8][9][10] and also for non-redox reactions,asinthe case of thiocytidine and thiouridine synthesis in tRNAs. [11][12][13][14] Thep resence of sulfur atoms within the cluster prompts to question whether the sulfur transferase could use these "internal" Sa toms as as ource of sulfur for the sulfuration reaction (Scheme 1A).…”

“…Such mechanism, which implies that the cluster is partly destroyed and repaired during catalysis,a nd thus that the enzyme cycles between a[4Fe-3S] state and an active [4Fe-4S] state (Scheme 1A), has been proposed for biotin or lipoate synthesis,c atalyzed by the BioB and LipA enzymes,r espectively. [7][8][9][10] In contrast, for thiouridine or thiocytidine synthesis during tRNAm odification, catalyzed by TtuA [12][13][14] and TtcA, [11] respectively,w ea nd others proposed am echanism Scheme 1. [4Fe-4S] clusters can either give or receive asulfur atom for sulfuration reactions.…”

The indispensable role of mammalian iron sulfur proteins in function and regulation of multiple diverse metabolic pathways

“…In some proteins such as lipoic acid synthase, which requires two Fe–S clusters, it is not yet clear exactly how other potential scaffolds participate in cluster transfer. They may mediate transfer as secondary scaffolds that contain targeting information for a specific subset and type of recipient proteins, such as those that contain Fe–S proteins that are consumed during incorporation of sulfur into octanoic acid to generate lipoic acid (6,8-dithiooctanoic acid), which functions in the swinging arm of multi- subunit dehydrogenases such as α-keto acid dehydrogenase, pyruvate dehydrogenase, branched chain ketoacid dehydrogenase and the glycine cleavage system (McCarthy and Booker 2017 ) (reviewed in Solmonson and DeBerardinis 2018 ) for which the exact molecular donor of the cluster is under study (McCarthy et al 2018 ). It is likely that multiple secondary scaffolds may acquire their Fe–S cofactors from the canonical Fe–S biogenesis machinery, and may then distribute their bound Fe–S to specific subsets of proteins based on specific protein recognition sites in recipient proteins that promote complex formation and enshrouded transfer of Fe–S clusters from the secondary scaffold.…”